Cole-parmer 8890 Manual
AbstractMat-forming benthic cyanobacteria are widespread throughout New Zealand rivers, and their ingestion has been linked to animal poisonings. In this study, potentially toxic benthic cyanobacterial proliferations were collected from 21 rivers and lakes throughout New Zealand.
Each environmental sample was screened for anatoxins using liquid chromatography-MS (LC-MS). Thirty-six cyanobacterial strains were isolated and cultured from these samples. A polyphasic approach was used to identify each isolate; this included genotypic analyses 16S rRNA gene sequences and intergenic spacer (ITS) and morphological characterization.
Each culture was analysed for anatoxins using LC-MS and screened for microcystin production potential using targeted PCR. The morphospecies Phormidium autumnale was found to be the dominant cyanobacterium in mat samples. Polyphasic analyses revealed multiple slight morphological variants within the P. Autumnale clade and highlighted the difficulties in identifying Oscillatoriaceae. Only one morphospecies (comprising the two strains CYN52 and CYN53) of P. Autumnale was found to produce anatoxins. These strains formed their own clade based on partial 16S rRNA gene sequences.
These data indicate that benthic P. Autumnale mats are composed of multiple morphospecies and toxin production is dependent on the presence of toxin-producing genotypes. Further cyanobacteria are also characterized, including Phormidium murrayi, which was identified for the first time outside of Antarctica. , IntroductionThe first report of toxin production in planktonic cyanobacteria was published in 1878. Since then, multiple incidents of animal and human poisonings have been linked to planktonic cyanobacteria and the cyanotoxins responsible have been identified. In contrast, there has been little information available on toxic benthic cyanobacteria.
Cole Parmer Barrington
However, over the past two decades, an increasing number of toxin-producing freshwater benthic cyanobacteria species have been documented (;;; ), and ingestion of these has been associated with animal poisonings (;; ). In New Zealand, reports of dog poisonings linked to benthic cyanobacteria have increased in the last 10 years. Since the first dog fatalities were documented in 1998 , there have been 30 reported deaths (, S.A. Wood, unpublished data.).Globally, anatoxins (neurotoxins) and microcystins (hepatotoxins) are the two most commonly produced cyanotoxins by benthic cyanobacteria (;;;; ).
Anatoxins are powerful neuromuscular-blocking agents that act through the nicotinic acetylcholine receptor, while microcystins inhibit protein phosphatases causing liver necrosis (; ). Recently, benthic cyanobacteria that produce cylindrospermopsins and saxitoxins have also been identified (; ). Saxitoxins are fast-acting neurotoxins that inhibit nerve conduction by blocking sodium channels; these toxins are common in marine dinoflagellates, where they are known as paralytic shellfish poisons,. Cylindrospermopsins are potent inhibitors of protein and glutathione synthesis acting on the liver and kidneys (; ). There have been no reported cases of animal toxicosis from benthic cyanobacteria producing saxitoxins and cylindrospermopsins. Benthic Phormidium and Oscillatoria sp.
Have most commonly been linked to cyanotoxin production (;;;; ). However, cyanotoxins have also been found in benthic species of Spirulina (microcystins and anatoxins), Fischerella (microcystins), Lyngbya (saxitoxins and cylindrospermopsins), Aphanothece (microcystins) and Nostoc (microcystins) (;;;; ).In New Zealand rivers, benthic mat-forming cyanobacteria are found under a wide range of water quality conditions. The most common mat-forming genus in New Zealand is Phormidium. Under optimal conditions, Phormidium forms expansive black/brown/green leathery mats over wide areas of river substrate. In the 2005/06 summer, identified the causative cyanobacterium of multiple dog deaths in the Hutt River (Wellington, New Zealand) as Phormidium autumnale.
This organism is the only benthic species known to produce anatoxin-a (ATX) and homoanatoxin-a (HTX) in New Zealand. Routine testing of Phormidium mats from around New Zealand has shown marked variations in the presence of anatoxins, in the anatoxin variants produced and in their concentrations.
There is uncertainty as to whether this variability is caused by the presence of different strains within the mats or to variations in their ability to produce anatoxins, or by environmental triggers, i.e. The correct and early identification of cyanobacterial species and confirmation of those species that produce toxins will provide guidance that can be used in developing management and mitigation programmes aimed at protecting animal and human health.In this study, 31 potentially toxic benthic proliferations of cyanobacteria were collected from 21 rivers and lakes around New Zealand. Each environmental sample was screened for anatoxins using liquid chromatography-MS (LC-MS). Individual isolates from each sample were cultured and anatoxins were analysed using LC-MS.
Each culture was screened for microcystin production potential using PCR. Isolates from the pure cultures were identified where possible to the species level using morphology and phylogenetic analyses. This study is the first to describe the diversity and toxin production of benthic cyanobacteria in New Zealand. Materials and methods Site description and sample collectionBetween 2005 and 2008, benthic cyanobacterial mats were collected from 21 New Zealand rivers and lakes experiencing cyanobacterial proliferations. Sampling sites from which cyanobacterial strains were successfully isolated are shown in. Cyanobacterial mats were predominantly found on rocky substrates, but were also collected from fine substrate (0.2–0.02 mm) in the Whakatikei and Rangitaiki rivers.
Samples collected from the Waikato river were from a small geothermal tributary. Samples were collected by scraping mats into sterile plastic screw-cap bottles (50 mL, Biolab, New Zealand).
All samples were placed on ice for transport. On arrival at the laboratory, samples were frozen immediately without cryopreservation (−20 °C) for later culturing and toxin analysis.
Subsamples (10 mL) were preserved using Lugol's Iodine for morphological identification. Locations of successfully isolated cyanobacterial cultures. Strain isolation and culture conditionsFrozen cyanobacteria samples were thawed and cyanobacterial strains were isolated by streaking on a solid MLA medium. One half of the Petri dish containing the streaked material was covered in black PVC. This helped in isolating single filaments as some benthic cyanobacteria are motile and move towards light.
After approximately 2 weeks, when filaments had moved across the Petri dishes, single filaments were isolated by micropipetting and transferred to 24-well plates containing 500 μL MLA medium per well. Filaments were washed repeatedly and incubated under standard conditions (100±20 μmol photons m −2 s −1; 16: 8 h light: dark; 18±1 °C, Contherm, 190 RHS, New Zealand). Cycloheximide (100 μg mL −1) was used in selected cultures to reduce eukaryotic growth (; ). Successfully isolated strains were maintained in 50-mL plastic bottles (Biolab) under the above conditions. Morphological identificationSubsamples of each cyanobacterial strain (in stationary phase) were identified by microscopy (Zeiss Photomicroscope II, Germany).
Photomicrographs were taken using a digital camera (Canon Powershot S3IS) and further processed in photoshop 7.0 (Adobe). Nomarski interference contrast microscopy was used in addition to bright-field microscopy to enhance the contrast in unstained samples. Species identifications were made primarily by reference to. Thirty measurements were made of vegetative cell lengths and widths for each isolate, and detailed observations of phenotypic characteristics were noted for 15 filaments. Isolation of DNA and molecular characterizationSubsamples (500 μL) from each culture were centrifuged using an Eppendorf microcentrifuge (15 000 g, 1 min) and the supernatant was removed by sterile pipetting.
And locationCell width (μm)Cell length (μm)Developed apical cellCross wallsAnatoxin production (mg kg −1 DW)Environmental, ATX/HTX production (μg kg −1 WW or mg kg −1 DW)Accession no.ID of nearest match (accession no.)% IDSample locationMorphotype AVUW1, Avalon duck pond6.0–7.82.4–5.4Conical/capitate/calyptraGranular––P. Autumnale;98E:2672100 N:5999665VUW2, Lake Henley Outlet6.6–7.82.4–3.6Conical/capitate/calyptraGranular––P. Autumnale;98E:2735925 N:6025095VUW3, Wainuiomata River4.8–8.41.8–3.6Conical/capitate/calyptra––P.
Autumnale;98E:2674410 N:5990890VUW4, Hutt River7.2–7.84.2–6.6Conical/capitate/calyptraGranular–HTX (4 mg kg −1 DW) dhATX (53 mg kg −1 DW) dhHTX (9 mg kg −1 DW)P. Autumnale;98E:2674410 N:6004090VUW5, Waingongoro River4.8–6.02.4–3.6Conical/capitate/calyptraGranular–––T. Autumnale;98N:6199175VUW7, Wainuiomata River6.0–6.63.0–4.8Conical/capitate/calyptraGranular––P. Autumnale;98E:2674410 N:5990890VUW9, Hutt River7.8–9.63.6–5.4Conical/capitate/calyptraGranular––P. Autumnale:98E:2689075 N:6010880VUW11, Pembroke road6.0–7.22.4–4.8Capitate/calyptra–NTP. Autumnale;98E:2657520 N:5991385VUW14, Hutt River7.2–8.42.4–3.6Conical/calyptraGranular–HTX (9 mg kg −1 DW) dhATX (325 mg kg −1 DW) dhHTX (95 mg kg −1 DW)P.
Autumnale;98E:2670240 N:5998870VUW16, Pelorous River6.6–7.82.4–4.2Conical/capitate/calyptraGranular––P. Autumnale;98E:2558175 N:5989825VUW17, Mangatinoka Stream6.0–7.83.0–4.2Conical/capitate/calyptraGranular––P.
Autumnale;98E:2752100 N:6082600VUW18, Makarewa River6.0–7.22.4–3.6Conical/capitate/calyptra––P. Autumnale;98E:2146875 N:5420325VUW19, Mangaroa River6.0–7.23.0–4.8Conical/capitate/calyptraGranular––P. Autumnale;98E:2688625 N:6010315VUW20, Rangataiki River6.6–8.43.6–4.8Conical/capitate/calyptraGranular––P. Autumnale;99E:2835500 N:6304300VUW21, Whakatane River7.2–8.43.0–5.4Conical/capitate/calyptraGranular––P. Bourrellyi;98N:6312550VUW22, Waimana River7.2–8.43.0–6.0Conical/capitate/calyptraGranular––P.
Autumnale;98E:2869700 N:6320350VUW23, Godley River Tributary6.6–8.43.0–4.8Conical/capitate/calyptraGranular––P. Bourrellyi;98N:5720475VUW24, Tukituki River6.0–9.63.0–4.8Conical/capitate/calyptraGranular––P. Autumnale;98CYN47, Ashley River6.0–7.23.0–6.0Conical/capitate/calyptraGranular–ATX/HTXP.
Autumnale;98E:2447450 N:5774580CYN48, Ashley River6.0–7.23.0–6.0Conical/capitate/calyptraGranular–ATX/HTXP. Autumnale;98E:2475800 N:5769700CYN49, Hutt River5.4–6.62.4–4.2Conical/capitate/calyptraGranular–ATX/HTXP.autumnale;98E:2670240 N:5998870CYN52, Rangataiki River4.2–6.03.0–5.4Conical/capitate/calyptraATX (1000 mg kg −1)ATX (200 mg kg −1 WW)T. Bourrellyi;99E:2845850 N:6350050CYN53, Rangataiki River4.2–6.03.0–5.4Conical/capitate/calyptraATX (1000 mg kg −1)ATX (200 mg kg −1 WW)T. Bourrellyi;99E:2845850 N:6350050CYN55, Roding River8.4–9.65.4–6.6Conical/capitate/calyptraGranular–NTP. Autumnale;99E:2523465 N:5977465Morphotype BVUW8, Akatarawa River9.6–13.21.8–4.2Conical/capitate/calyptra–Trace levelsP. Autumnale;99E:2686195 N:6010975VUW10, Wainuiomata River8.4–122.4–4.8Thicken/calyptraGranular–NTP. Autumnale;99E:2678253 N:5992345VUW12, Wainuiomata River9.6–122.4–4.2Thicken/calyptraGranular––P.
Autumnale;99E:2678253 N:5992345Morphotype CVUW30, Waikato River9.0–15.61.2–4.2RoundedGranular–––E:2776835Slightly restrictedN:6278400Morphotype DCYN38, Red Hills Tarn3.6–4.22.4–4.2Rounded/conicalSlightly granular–NTP. Murrayi;98E:251330098N:5952770CYN39, Red Hills Tarn3.6–4.22.4–4.2Rounded/conicalSlightly granular–NTP. Murrayi;98E:2513300 N:5952770Morphotype EVUW13, Mangataurhiri Stream3.6–4.83.6–4.8Rounded––Symploca sp.;94E:2701830M.paludosus;93N:6454770M. And locationCell width (μm)Cell length (μm)Developed apical cellCross wallsAnatoxin production (mg kg −1 DW)Environmental, ATX/HTX production (μg kg −1 WW or mg kg −1 DW)Accession no.ID of nearest match (accession no.)% IDSample locationMorphotype AVUW1, Avalon duck pond6.0–7.82.4–5.4Conical/capitate/calyptraGranular––P.
Autumnale;98E:2672100 N:5999665VUW2, Lake Henley Outlet6.6–7.82.4–3.6Conical/capitate/calyptraGranular––P. Autumnale;98E:2735925 N:6025095VUW3, Wainuiomata River4.8–8.41.8–3.6Conical/capitate/calyptra––P. Autumnale;98E:2674410 N:5990890VUW4, Hutt River7.2–7.84.2–6.6Conical/capitate/calyptraGranular–HTX (4 mg kg −1 DW) dhATX (53 mg kg −1 DW) dhHTX (9 mg kg −1 DW)P. Autumnale;98E:2674410 N:6004090VUW5, Waingongoro River4.8–6.02.4–3.6Conical/capitate/calyptraGranular–––T. Autumnale;98N:6199175VUW7, Wainuiomata River6.0–6.63.0–4.8Conical/capitate/calyptraGranular––P. Autumnale;98E:2674410 N:5990890VUW9, Hutt River7.8–9.63.6–5.4Conical/capitate/calyptraGranular––P.
Autumnale:98E:2689075 N:6010880VUW11, Pembroke road6.0–7.22.4–4.8Capitate/calyptra–NTP. Autumnale;98E:2657520 N:5991385VUW14, Hutt River7.2–8.42.4–3.6Conical/calyptraGranular–HTX (9 mg kg −1 DW) dhATX (325 mg kg −1 DW) dhHTX (95 mg kg −1 DW)P. Autumnale;98E:2670240 N:5998870VUW16, Pelorous River6.6–7.82.4–4.2Conical/capitate/calyptraGranular––P. Autumnale;98E:2558175 N:5989825VUW17, Mangatinoka Stream6.0–7.83.0–4.2Conical/capitate/calyptraGranular––P. Autumnale;98E:2752100 N:6082600VUW18, Makarewa River6.0–7.22.4–3.6Conical/capitate/calyptra––P.
Autumnale;98E:2146875 N:5420325VUW19, Mangaroa River6.0–7.23.0–4.8Conical/capitate/calyptraGranular––P. Autumnale;98E:2688625 N:6010315VUW20, Rangataiki River6.6–8.43.6–4.8Conical/capitate/calyptraGranular––P. Autumnale;99E:2835500 N:6304300VUW21, Whakatane River7.2–8.43.0–5.4Conical/capitate/calyptraGranular––P. Bourrellyi;98N:6312550VUW22, Waimana River7.2–8.43.0–6.0Conical/capitate/calyptraGranular––P. Autumnale;98E:2869700 N:6320350VUW23, Godley River Tributary6.6–8.43.0–4.8Conical/capitate/calyptraGranular––P.
Bourrellyi;98N:5720475VUW24, Tukituki River6.0–9.63.0–4.8Conical/capitate/calyptraGranular––P. Autumnale;98CYN47, Ashley River6.0–7.23.0–6.0Conical/capitate/calyptraGranular–ATX/HTXP. Autumnale;98E:2447450 N:5774580CYN48, Ashley River6.0–7.23.0–6.0Conical/capitate/calyptraGranular–ATX/HTXP.
Autumnale;98E:2475800 N:5769700CYN49, Hutt River5.4–6.62.4–4.2Conical/capitate/calyptraGranular–ATX/HTXP.autumnale;98E:2670240 N:5998870CYN52, Rangataiki River4.2–6.03.0–5.4Conical/capitate/calyptraATX (1000 mg kg −1)ATX (200 mg kg −1 WW)T. Bourrellyi;99E:2845850 N:6350050CYN53, Rangataiki River4.2–6.03.0–5.4Conical/capitate/calyptraATX (1000 mg kg −1)ATX (200 mg kg −1 WW)T. Bourrellyi;99E:2845850 N:6350050CYN55, Roding River8.4–9.65.4–6.6Conical/capitate/calyptraGranular–NTP. Autumnale;99E:2523465 N:5977465Morphotype BVUW8, Akatarawa River9.6–13.21.8–4.2Conical/capitate/calyptra–Trace levelsP. Autumnale;99E:2686195 N:6010975VUW10, Wainuiomata River8.4–122.4–4.8Thicken/calyptraGranular–NTP.
Autumnale;99E:2678253 N:5992345VUW12, Wainuiomata River9.6–122.4–4.2Thicken/calyptraGranular––P. Autumnale;99E:2678253 N:5992345Morphotype CVUW30, Waikato River9.0–15.61.2–4.2RoundedGranular–––E:2776835Slightly restrictedN:6278400Morphotype DCYN38, Red Hills Tarn3.6–4.22.4–4.2Rounded/conicalSlightly granular–NTP. Murrayi;98E:251330098N:5952770CYN39, Red Hills Tarn3.6–4.22.4–4.2Rounded/conicalSlightly granular–NTP. Murrayi;98E:2513300 N:5952770Morphotype EVUW13, Mangataurhiri Stream3.6–4.83.6–4.8Rounded––Symploca sp.;94E:2701830M.paludosus;93N:6454770M. And locationCell width (μm)Cell length (μm)Developed apical cellCross wallsAnatoxin production (mg kg −1 DW)Environmental, ATX/HTX production (μg kg −1 WW or mg kg −1 DW)Accession no.ID of nearest match (accession no.)% IDSample locationMorphotype AVUW1, Avalon duck pond6.0–7.82.4–5.4Conical/capitate/calyptraGranular––P.
Autumnale;98E:2672100 N:5999665VUW2, Lake Henley Outlet6.6–7.82.4–3.6Conical/capitate/calyptraGranular––P. Autumnale;98E:2735925 N:6025095VUW3, Wainuiomata River4.8–8.41.8–3.6Conical/capitate/calyptra––P. Autumnale;98E:2674410 N:5990890VUW4, Hutt River7.2–7.84.2–6.6Conical/capitate/calyptraGranular–HTX (4 mg kg −1 DW) dhATX (53 mg kg −1 DW) dhHTX (9 mg kg −1 DW)P. Autumnale;98E:2674410 N:6004090VUW5, Waingongoro River4.8–6.02.4–3.6Conical/capitate/calyptraGranular–––T. Autumnale;98N:6199175VUW7, Wainuiomata River6.0–6.63.0–4.8Conical/capitate/calyptraGranular––P. Autumnale;98E:2674410 N:5990890VUW9, Hutt River7.8–9.63.6–5.4Conical/capitate/calyptraGranular––P. Autumnale:98E:2689075 N:6010880VUW11, Pembroke road6.0–7.22.4–4.8Capitate/calyptra–NTP.
Autumnale;98E:2657520 N:5991385VUW14, Hutt River7.2–8.42.4–3.6Conical/calyptraGranular–HTX (9 mg kg −1 DW) dhATX (325 mg kg −1 DW) dhHTX (95 mg kg −1 DW)P. Autumnale;98E:2670240 N:5998870VUW16, Pelorous River6.6–7.82.4–4.2Conical/capitate/calyptraGranular––P.
Autumnale;98E:2558175 N:5989825VUW17, Mangatinoka Stream6.0–7.83.0–4.2Conical/capitate/calyptraGranular––P. Autumnale;98E:2752100 N:6082600VUW18, Makarewa River6.0–7.22.4–3.6Conical/capitate/calyptra––P. Autumnale;98E:2146875 N:5420325VUW19, Mangaroa River6.0–7.23.0–4.8Conical/capitate/calyptraGranular––P. Autumnale;98E:2688625 N:6010315VUW20, Rangataiki River6.6–8.43.6–4.8Conical/capitate/calyptraGranular––P. Autumnale;99E:2835500 N:6304300VUW21, Whakatane River7.2–8.43.0–5.4Conical/capitate/calyptraGranular––P. Bourrellyi;98N:6312550VUW22, Waimana River7.2–8.43.0–6.0Conical/capitate/calyptraGranular––P. Autumnale;98E:2869700 N:6320350VUW23, Godley River Tributary6.6–8.43.0–4.8Conical/capitate/calyptraGranular––P.
Bourrellyi;98N:5720475VUW24, Tukituki River6.0–9.63.0–4.8Conical/capitate/calyptraGranular––P. Autumnale;98CYN47, Ashley River6.0–7.23.0–6.0Conical/capitate/calyptraGranular–ATX/HTXP. Autumnale;98E:2447450 N:5774580CYN48, Ashley River6.0–7.23.0–6.0Conical/capitate/calyptraGranular–ATX/HTXP. Autumnale;98E:2475800 N:5769700CYN49, Hutt River5.4–6.62.4–4.2Conical/capitate/calyptraGranular–ATX/HTXP.autumnale;98E:2670240 N:5998870CYN52, Rangataiki River4.2–6.03.0–5.4Conical/capitate/calyptraATX (1000 mg kg −1)ATX (200 mg kg −1 WW)T.
Bourrellyi;99E:2845850 N:6350050CYN53, Rangataiki River4.2–6.03.0–5.4Conical/capitate/calyptraATX (1000 mg kg −1)ATX (200 mg kg −1 WW)T. Bourrellyi;99E:2845850 N:6350050CYN55, Roding River8.4–9.65.4–6.6Conical/capitate/calyptraGranular–NTP. Autumnale;99E:2523465 N:5977465Morphotype BVUW8, Akatarawa River9.6–13.21.8–4.2Conical/capitate/calyptra–Trace levelsP. Autumnale;99E:2686195 N:6010975VUW10, Wainuiomata River8.4–122.4–4.8Thicken/calyptraGranular–NTP.
Autumnale;99E:2678253 N:5992345VUW12, Wainuiomata River9.6–122.4–4.2Thicken/calyptraGranular––P. Autumnale;99E:2678253 N:5992345Morphotype CVUW30, Waikato River9.0–15.61.2–4.2RoundedGranular–––E:2776835Slightly restrictedN:6278400Morphotype DCYN38, Red Hills Tarn3.6–4.22.4–4.2Rounded/conicalSlightly granular–NTP. Murrayi;98E:251330098N:5952770CYN39, Red Hills Tarn3.6–4.22.4–4.2Rounded/conicalSlightly granular–NTP. Murrayi;98E:2513300 N:5952770Morphotype EVUW13, Mangataurhiri Stream3.6–4.83.6–4.8Rounded––Symploca sp.;94E:2701830M.paludosus;93N:6454770M.
And locationCell width (μm)Cell length (μm)Developed apical cellCross wallsAnatoxin production (mg kg −1 DW)Environmental, ATX/HTX production (μg kg −1 WW or mg kg −1 DW)Accession no.ID of nearest match (accession no.)% IDSample locationMorphotype AVUW1, Avalon duck pond6.0–7.82.4–5.4Conical/capitate/calyptraGranular––P. Autumnale;98E:2672100 N:5999665VUW2, Lake Henley Outlet6.6–7.82.4–3.6Conical/capitate/calyptraGranular––P. Autumnale;98E:2735925 N:6025095VUW3, Wainuiomata River4.8–8.41.8–3.6Conical/capitate/calyptra––P. Autumnale;98E:2674410 N:5990890VUW4, Hutt River7.2–7.84.2–6.6Conical/capitate/calyptraGranular–HTX (4 mg kg −1 DW) dhATX (53 mg kg −1 DW) dhHTX (9 mg kg −1 DW)P. Autumnale;98E:2674410 N:6004090VUW5, Waingongoro River4.8–6.02.4–3.6Conical/capitate/calyptraGranular–––T.
Autumnale;98N:6199175VUW7, Wainuiomata River6.0–6.63.0–4.8Conical/capitate/calyptraGranular––P. Autumnale;98E:2674410 N:5990890VUW9, Hutt River7.8–9.63.6–5.4Conical/capitate/calyptraGranular––P. Autumnale:98E:2689075 N:6010880VUW11, Pembroke road6.0–7.22.4–4.8Capitate/calyptra–NTP. Autumnale;98E:2657520 N:5991385VUW14, Hutt River7.2–8.42.4–3.6Conical/calyptraGranular–HTX (9 mg kg −1 DW) dhATX (325 mg kg −1 DW) dhHTX (95 mg kg −1 DW)P. Autumnale;98E:2670240 N:5998870VUW16, Pelorous River6.6–7.82.4–4.2Conical/capitate/calyptraGranular––P. Autumnale;98E:2558175 N:5989825VUW17, Mangatinoka Stream6.0–7.83.0–4.2Conical/capitate/calyptraGranular––P. Autumnale;98E:2752100 N:6082600VUW18, Makarewa River6.0–7.22.4–3.6Conical/capitate/calyptra––P.
Autumnale;98E:2146875 N:5420325VUW19, Mangaroa River6.0–7.23.0–4.8Conical/capitate/calyptraGranular––P. Autumnale;98E:2688625 N:6010315VUW20, Rangataiki River6.6–8.43.6–4.8Conical/capitate/calyptraGranular––P. Autumnale;99E:2835500 N:6304300VUW21, Whakatane River7.2–8.43.0–5.4Conical/capitate/calyptraGranular––P.
Bourrellyi;98N:6312550VUW22, Waimana River7.2–8.43.0–6.0Conical/capitate/calyptraGranular––P. Autumnale;98E:2869700 N:6320350VUW23, Godley River Tributary6.6–8.43.0–4.8Conical/capitate/calyptraGranular––P. Bourrellyi;98N:5720475VUW24, Tukituki River6.0–9.63.0–4.8Conical/capitate/calyptraGranular––P. Autumnale;98CYN47, Ashley River6.0–7.23.0–6.0Conical/capitate/calyptraGranular–ATX/HTXP. Autumnale;98E:2447450 N:5774580CYN48, Ashley River6.0–7.23.0–6.0Conical/capitate/calyptraGranular–ATX/HTXP.
Autumnale;98E:2475800 N:5769700CYN49, Hutt River5.4–6.62.4–4.2Conical/capitate/calyptraGranular–ATX/HTXP.autumnale;98E:2670240 N:5998870CYN52, Rangataiki River4.2–6.03.0–5.4Conical/capitate/calyptraATX (1000 mg kg −1)ATX (200 mg kg −1 WW)T. Bourrellyi;99E:2845850 N:6350050CYN53, Rangataiki River4.2–6.03.0–5.4Conical/capitate/calyptraATX (1000 mg kg −1)ATX (200 mg kg −1 WW)T. Bourrellyi;99E:2845850 N:6350050CYN55, Roding River8.4–9.65.4–6.6Conical/capitate/calyptraGranular–NTP. Autumnale;99E:2523465 N:5977465Morphotype BVUW8, Akatarawa River9.6–13.21.8–4.2Conical/capitate/calyptra–Trace levelsP.
Autumnale;99E:2686195 N:6010975VUW10, Wainuiomata River8.4–122.4–4.8Thicken/calyptraGranular–NTP. Autumnale;99E:2678253 N:5992345VUW12, Wainuiomata River9.6–122.4–4.2Thicken/calyptraGranular––P. Autumnale;99E:2678253 N:5992345Morphotype CVUW30, Waikato River9.0–15.61.2–4.2RoundedGranular–––E:2776835Slightly restrictedN:6278400Morphotype DCYN38, Red Hills Tarn3.6–4.22.4–4.2Rounded/conicalSlightly granular–NTP.
Murrayi;98E:251330098N:5952770CYN39, Red Hills Tarn3.6–4.22.4–4.2Rounded/conicalSlightly granular–NTP. Murrayi;98E:2513300 N:5952770Morphotype EVUW13, Mangataurhiri Stream3.6–4.83.6–4.8Rounded––Symploca sp.;94E:2701830M.paludosus;93N:6454770M. Phylogenetic tree of the Phormidium autumnale group based on the 16S rRNA gene sequences (647 bp) and obtained using the neighbour-joining method. Bootstrap values 50% are noted at the nodes.
The different morphotypes in this study are in bold. The remainder of the tree is shown in.To assess the microcystin-producing potential of each culture, amplification of a region of the mcyE gene was performed as described above using the HEPF and HEPR primers. PCR products were visualized on a 1.5% agarose gel.
Sequence alignment and phylogenetic analysisThe 16S rRNA gene sequences and ITS sequences were aligned using clustal w in mega 4. Pair-wise distances were calculated using the Jukes–Cantor method and pairwise deletion used to account for sequence length variation or gaps. Phylogenetic trees were constructed using a neighbour-joining algorithm and Tamura–Nei distance estimates. Bootstrap analyses of 1000 iterations were performed to identify the node support for the consensus trees.
Anatoxins analysisSubsamples of all cyanobacterial strains were lyophilized (FreeZone6, Labconco). Lyophilized material (100 mg) was resuspended in 10 mL of double-distilled water (DDW) containing 0.1% formic acid and sonicated (Cole Parmer 8890, Biolab) for 15 min. Samples were centrifuged at 4000 g for 10 min. The procedure was repeated using 5 mL DDW and the supernatants combined.All cyanobacterial strains were analysed for ATX, HTX and their degradation products using LC-MS.
Anatoxins were separated by LC (Acquity UPLC, Waters Corp., MA) using a 50′ 1-mm Acquity BEH-C18 (1.7 μm) column (Waters Corp.). Both the mobile phase A (water) and mobile phase B (acetonitrile) contained 0.1% formic acid and were used at a flow of 0.3 mL min −1, isocratic for 1 min at 100% A, followed by a rapid gradient from 100% A to 50% A/50% B over 2 min. The injection volume was 5 μL. The Quattro Premier XE mass spectrometer (Waters-Micromass, Manchester) was operated in the ESI+ mode with capillary voltage 0.5 kV, desolvation gas 900 L h −1, 400 °C, cone gas 200 L h −1 and cone voltage 25 V.
Quantitative analysis was performed by multiple-reaction monitoring using MS-MS channels set up for ATX (166.15149.1; R t 1.0 min), HTX (180.2163.15; R t c. 1.9 min), dihydroanatoxin-a (168.156; R t 0.9 min), dihydrohomoanatoxin-a (182.157; R t c.
1.9 min), epoxyanatoxin-a (182.198) and epoxyhomoanatoxin (196.1140; R t c. The instrument was calibrated with dilutions in 0.1% formic acid of authentic standards of ATX (A.G. Scientific, CA). Results Environmental samples and strain isolationThe majority of the 31 cyanobacterial mats sampled were collected from black/green/brown leathery mats consistent with that described for Phormidium (; ). Only two other mat types were found and collected: green gelatinous Nostoc colonies and one brittle brown filamentous mat.
Preliminary microscopic observation of the leathery mats confirmed that they were comprised almost entirely of filamentous Phormidium sp. Representatives of Oscillatoria were observed among two of the Phormidium-dominated mats. Additionally, Pseudanabaenaceae were observed in at least 10 of the samples, but these species proved difficult to isolate and culture.
The brittle brown mat was a proliferation of two species from the Pseudanabaenaceae. Of the Nostoc colonies observed, only one could be cultured and described. This strain of Nostoc was found to be growing with an Oscillatoria sp.; together, they were the only species collected from a geothermal location. Thirty-six unicellular cyanobacterial strains were successfully isolated and on-grown. Within all mat samples, a mixture of diatoms was also observed.
Melosira, Cymbella, Frustulia and Gomphonema were the most prominent. Microscopic characterization of isolatesMorphological characteristics including cell dimensions, apical cell profile, cross wall configuration and reproductive structures for each isolate are given in. All isolates were found to have traits in common with Oscillatoriales, with the exception of VUW31, which was placed in the Nostocales.Cells from the majority of isolates were characterized by being isodiametric or slightly shorter than wide.
Cells were generally 4–8.5 μm wide. Trichomes were motile and straight with well-defined apical cells with a calyptra (thickened membrane).
Those isolates sharing these morphological characteristics were identified as P. Autumnale (Agardh) Trevisan ex Gomont 1892 (;; ) and assigned to Morphotype A (, ).
Cole Parmer Catalog
Twenty-four strains VUW1–5, 7, 9, 11, 14, 16–24, CYN47–49, CYN52–53 and 55 were included in this designation. Morphological variation was observed among strains in apical cell morphology, cell-wall granulation, sheath presence or absence and thickness. Photomicrographs of nine different Phormidium autumnale strains, (a–h) Morphotype A and (i) Morphotype B. Photomicrographs images are all bright field, with the exception of (c) and (d), which were taken using Nomarski optics.
Cole Parmer Uk
(a) VUW18 Makarewa River, (b) VUW9 Hutt River, (c) VUW19 Mangaroa River, (d) CYN55 Roding River, (e) CYN53 Rangataiki River, (f) VUW1 Avalon Duck Pond, (g) VUW17 Mangatinoka Stream (Lugol's preserved), (h) VUW24 Tukituki River and (i) VUW10 Wainuiomata River (red border, only representative from Morphotype B). All are described in. Scale bars=20 μm. Photomicrographs of nine different Phormidium autumnale strains, (a–h) Morphotype A and (i) Morphotype B.
Photomicrographs images are all bright field, with the exception of (c) and (d), which were taken using Nomarski optics. (a) VUW18 Makarewa River, (b) VUW9 Hutt River, (c) VUW19 Mangaroa River, (d) CYN55 Roding River, (e) CYN53 Rangataiki River, (f) VUW1 Avalon Duck Pond, (g) VUW17 Mangatinoka Stream (Lugol's preserved), (h) VUW24 Tukituki River and (i) VUW10 Wainuiomata River (red border, only representative from Morphotype B). All are described in. Scale bars=20 μm.Stains VUW8, 10 and 12 were morphologically similar to strains from Morphotype A; however, the cell structure was discoid (width, 8.4–13.2 μm; length, 1.8–4.8 μm) and each had a distinctive, rounded to hemispherical calyptra. Isolates were identified as belonging to the genus Oscillatoria possessing a distinctly thickened apical cell with calyptra, no constrictions at cross walls and incomplete cross-wall formation during cell division (the main generic feature). These three strains were assigned to Morphotype B.
The only other strain identified as Oscillatoria was VUW30. This strain (Morphotype C) was distinctively granular, generally straight, possessing a discoid cell structure (width, 8.4–15.6 μm; length, 2.2–3.8 μm) and was slightly constricted at cross walls. Photomicrographs of four strains from contrasting genera. (a) VUW30 Waikato River (Morphotype C), (b) CYN38 Red Hills Tarn (Morphotype D), (c) VUW6 Wainuiomata River (Morphotype G) and (d) VUW31 Waikato River (Morphotype J). Scale bars=20 μm.
All images are bright field.Morphotype D was represented by strains CYN38 and 39, which were assigned to Phormidium murrayi (West and West 1911) (; ), with narrow, isodiametric cells (width, 3.6–4.2 μm; length, 2.4–4.2 μm), a prominent sheath (not always present) and conical/rounded apical cells with no calyptra.Strain VUW13 (Morphotype E) unfortunately stopped growing in culture before a full taxonomic classification could be provided; however, preliminary identification assigned this to the Phormidiaceae. It was distinctly different from all the other morphotypes.Single strains from the family Pseudanabaenaceae were assigned as Morphotypes F, G, H and I, and all differed slightly in morphology. Strains VUW6 (Morphotype F, ) and VUW28 (Morphotype I) were assigned to genus Leptolyngbya. Morphotype F was found in tightly tangled mats.
Trichomes were slightly constricted at cross walls. Cells were isodiametric (width, 1.8–2.4 μm; length, 1.2–2.4 μm). Apical cells were rounded/conical and sheath was absent.
Strain VUW28 (Morphotype I) formed a dense tangled mat, possessing no aerotopes, having a slight constriction, sheath sometimes present and a conical apical cell. Strain VUW15 (Morphotype H) was the only species isolated from the brittle brown mat. The strain was found to form benthic mats, comprising aerotopes at the septa, with a slight constriction at cross walls and cells two to three times longer than wide.
Strain VUW26 (Mophotype H) was found to be from the Pseudanabaena genus, defined by very brittle filaments and cell wall constriction. It formed a very loose ‘soup’ of filaments.Finally, strain VUW31 was the only representative of Nostoc.
Possessing distinctive akinetes and heterocytes, this strain was identified as Nostoc muscorum and designated as Morphotype J. Genotypic analyses and comparison with morphological designationsThe 16S rRNA gene sequences (647 bp) for all 36 cultures, with the exception of culture VUW30, were used to construct a neighbour-joining tree to determine the phylogenetic relationship between isolates and other cyanobacterial 16S rRNA gene sequences obtained from GenBank ( and ). Phylogenetic tree based on the 16S rRNA gene sequences (647 bp) and obtained using the neighbour-joining method.
Bootstrap values 50% are noted at the nodes. The different morphotypes from this study are in bold. The Phormidium autumnale section of the tree is shown in.Twenty-seven of the cultured isolates clustered together with 100% bootstrap support. Using blastn, all 27 sequences in this group matched at 98% sequence homology with P. Autumnale representatives from GenBank (all GenBank representatives were from peer-reviewed publications). The 27 isolates, however, shared a sequence similarity 97% sequence similarity (; ). Other strains on the sister branch were distant, sharing 98% sequence similarity.Finally, strain VUW31 (Morphotype J) was found to cluster with other heterocytous species and had 99% node support with Nostoc moscorum.ITS sequences (727 bp) were successfully obtained for 12 isolates from within the Phormidium clade (VUW 2, 4, 8, 9, 16–18, 24, CYN 47–49 and 55).
These sequences were used to construct a neighbour-joining tree. In general, the phylogenetic relationships observed in this tree matched those observed in the phylogenetic analysis of 16S rRNA gene sequences (data not shown).
The ITS sequences did not provide any additional information to allow finer scale resolution of intraspecies identification within the P. Autumnale clade. Cyanotoxin analysisLC-MS analysis identified anatoxins in seven of the 31 environmental samples.
All seven mats were dominated by Phormidium. Interestingly, ATX was detected in only two of the 36 strains (CYN52 and CYN53), while HTX and its degradation products were not detected. Both strains produced ATX at 1000 mg kg −1 freeze-dried weight. Both strains CYN52 and CYN53 were isolated from the same environmental sample sourced from the Rangataiki River and identified by morphology and phylogenetic analysis as P. These two strains also formed their own clade in the phylogenetic tree sharing 100% sequence similarity.No isolates were found to contain the mcyE gene, with only the positive control testing positive.All strains isolated in this study have been cryopreserved successfully using the methods of, and these are banked and maintained in the Cawthron Institute culture collection. DiscussionMorphological investigation revealed the presence of nine different morphotypes, all from the Oscillatoriales and one (Morphotype J) from the Nostocales. Morphotypes A and BPhormidium autumnale is known to have a cosmopolitan distribution and has been identified in many different habitats (; ).
It has unusually broad morphological and physiological characteristics. These features were also observed in this study. Morphotype A consisted of 24 isolates that were identified as P. Autumnale by polyphasic assessment. These 24 isolates could not be further separated based on morphological criteria. Considerable morphological variation was, however, observed between isolates, but this variation was confined within the broad P.
Autumnale definition. Cell granulation, apical cell profile and sheath presence were all found to vary. Sheath production, traditionally used for the systematic classification of Oscillatorialean (;; ), has been shown to be subject to the direct effects of both environmental and culture conditions (; ). Apical cell structures (in particular, the calyptra) vary in trichomes of different ages and are rarely seen in culture. The observed variation seen in this study is in contrast to, who demonstrated that 10 P.
Autumnale ssp. Exhibited a relatively similar morphology in culture.Intraspecific identification was therefore not possible in this group by morphology. However, the morphological heterogeneity observed in Morphotype A was represented by a number of genotype variations in the phylogenetic analysis.
These genotype variations resulted in the formation of 10 different clades, indicating the presence of subspecies within this morphotype. This is consistent with, who were unable to separate their P. Autumnale strains Arct-Ph5 and Ant-Ph68 based on morphological characteristics, but identified two subspecies by genetic analysis (16S rRNA gene sequence). Strains CYN52 and 53 formed their own clade within Morphotype A and were the only anatoxin producers in this lineage. Isolation of further anatoxin-producing strains is required to determine whether this divergence is consistent for all anatoxin producers.Unexpectedly, the three strains of Morphotype B, identified as Oscillatoria by morphology, formed their own clade within this P. Autumnale lineage. Morphotype B has disc-like cells and a hemispherical calyptra.
These features contrast with those of Morphotype A. Found that cell size varies not only between the strains but also within the strains of P. This was also found in the current study, where Morphotype B possessed cell widths ranging between 8.4 and 13.2 μm. This is larger and with a greater variation than that described previously for this species and differs markedly from Morphotype A. Apical cell structure (calyptra) furthermore has been recognized as a stable morphological structure used for distinguishing species. In this study, two distinct calyptra types were observed among Morphotypes A and B , indicating that these two morphotypes should be maintained as two distinct species.The phylogenetic analysis (16S rRNA gene sequences) showed that the P.
Autumnale lineage consisted of all isolates from Morphotypes A and B. Additionally, GenBank representatives Microcoleus sp.
And Tychonema bourrellyi were included in this clade. This clade was supported by 99% bootstrap support; however, together, they shared 97.5% as a threshold for bacterial species definition, while 95% has been used as a genus barrier for the 16S rRNA gene (;; ).
This standard led to conclude that their two morphologically similar P. Autumnale groups that shared.
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